Makoto Nakamura

Development of a three-dimensional bioprinter: construction of cell supporting structures using hydrogel and state-of-the-art inkjet technology.

We have developed a new technology for producing three-dimensional (3D) biological structures composed of living cells and hydrogel in vitro, via the direct and accurate printing of cells with an inkjet printing system. Various hydrogel structures were constructed with our custom-made inkjet printer, which we termed 3D bioprinter. In the present study, we used an alginate hydrogel that was obtained through the reaction of a sodium alginate solution with a calcium chloride solution. For the construction of the gel structure, sodium alginate solution was ejected from the inkjet nozzle (SEA-Jet, Seiko Epson Corp., Suwa, Japan) and was mixed with a substrate composed of a calcium chloride solution. In our 3D bioprinter, the nozzle head can be moved in three dimensions. Owing to the development of the 3D bioprinter, an innovative fabrication method that enables the gentle and precise fixation of 3D gel structures was established using living cells as a material. To date, several 3D structures that include living cells have been fabricated, including lines, planes, laminated structures, and tubes, and now, experiments to construct various hydrogel structures are being carried out in our laboratory.

Biofabrication: reappraising the definition of an evolving field

Biofabrication is an evolving research field that has recently received significant attention. In particular, the adoption of Biofabrication concepts within the field of Tissue Engineering and Regenerative Medicine has grown tremendously, and has been accompanied by a growing inconsistency in terminology. This article aims at clarifying the position of Biofabrication as a research field with a special focus on its relation to and application for Tissue Engineering and Regenerative Medicine. Within this context, we propose a refined working definition of Biofabrication, including Bioprinting and Bioassembly as complementary strategies within Biofabrication.

Three-dimensional inkjet biofabrication based on designed images

Tissue engineering has been developed with the ultimate aim of manufacturing human organs, but success has been limited to only thin tissues and tissues with no significant structures. In order to construct more complicated tissues, we have developed a three-dimensional (3D) fabrication technology in which 3D structures are directly built up by layer-by-layer printing with living cells and several tissue components. We developed a custom-made inkjet printer specially designed for this purpose. Recently, this printer was improved, and the on-demand printing mode was developed and installed to fabricate further complicated structures. As a result of this version, 3D layer-by-layer printing based on complicated image data has become possible, and several 2D and 3D structures with more complexity than before were successfully fabricated. The effectiveness of the on-demand printing mode in the fabrication of complicated 3D tissue structures was confirmed. As complicated 3D structures are essential for biofunctional tissues, inkjet 3D biofabrication has great potential for engineering complicated bio-functional tissues.

Bioprinting is coming of age: report from the International Conference on Bioprinting and Biofabrication in Bordeaux (3B'09)

The International Conference on Bioprinting and Biofabrication in Bordeaux (3B09) demonstrated that the field of bioprinting and biofabrication continues to evolve. The increasing number and broadening geography of participants, the emergence of new exciting bioprinting technologies, and the attraction of young investigators indicates the strong growth potential of this emerging field. Bioprinting can be defined as the use of computer-aided transfer processes for patterning and assembling living and non-living materials with a prescribed 2D or 3D organization in order to produce bio-engineered structures serving in regenerative medicine, pharmacokinetic and basic cell biology studies. The use of bioprinting technology for biofabrication of in vitro assay has been shown to be a realistic short-term application. At the same time, the principal feasibility of bioprinting vascularized human organs as well as in vivo bioprinting has been demonstrated. The bioprinting of complex 3D human tissues and constructs in vitro and especially in vivo are exciting, but long-term, applications. It was decided that the 5th International Conference on Bioprinting and Biofabrication would be held in Philadelphia, USA in October 2010. The specially appointed Eploratory Committee will consider the possibility of turning the growing bioprinting community into a more organized entity by creating a new bioprinting and biofabrication society. The new journal Biofabrication was also presented at 3B09. This is an important milestone per se which provides additional objective evidence that the bioprinting and biofabrication field is consolidating and maturing. Thus, it is safe to state that bioprinting technology is coming of age.

Bio rapid prototyping by extruding/aspirating/refilling thermoreversible hydrogel

This paper reports a method for rapid prototyping of cell tissues, which is based on a system that extrudes, aspirates and refills a mixture of cells and thermoreversible hydrogel as a scaffold. In the extruding mode, a cell-mixed scaffold solution in the sol state is extruded from a cooled micronozzle into a temperature-controlled substrate, which keeps the scaffold in the gel state. In the aspiration mode, the opposite process is performed by Bernoulli suction. In the refilling mode, the solution is extruded into a groove created in the aspiration mode. The minimum width of extruded hydrogel pattern is 114 +/- 15 microm by employing a nozzle of diameter 100 microm, and that of aspirated groove was 355 +/- 10 microm using a 500 microm-diameter nozzle. Gum arabic is mixed with the scaffold solution to avoid peeling-off of the gel pattern from the substrate. Patterning of Sf-9 cell tissue is demonstrated, and the stability of the patterned cell is investigated. This system offers a procedure for rapid prototyping and local modification of cell scaffolds for tissue engineering.

Anti-biofouling properties of an amphoteric polymer brush constructed on a glass substrate.

An amphoteric copolymer brush of methacrylic acid (MA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) was prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization using both a free chain transfer agent (n-butylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid) and a radical initiator (4,4-azobis(4-cyanopentanoic acid)) covalently fixed to a glass substrate. An aqueous solution of the copolymer, Poly(MA-r-DMAEMA), which was simultaneously obtained in liquid phase, had a sufficiently small polydispersity in its molecular weight. The copolymer brush showed effective suppression of non-specific adsorption of bovine serum albumin and egg white lysozyme to the brush. In contrast, both negatively charged PolyMA and positively charged PolyDMAEMA brushes significantly adsorbed the proteins irrespective of their net charges. Upon ion beam irradiation, furthermore, a hollow space with a designed shape could be made on the glass substrate, and both HEK293 and HepG2 cells non-specifically adhered to the space, forming aggregates, while no adhesion to the non-treated area on the brush was observed. These results suggest that the amphoteric polymer brushes will be useful materials for biomedical applications.

Image printing on the surface of anti-biofouling zwitterionic polymer brushes by ion beam irradiation.

A CMB monomer was polymerized on a glass plate with a surface-confined ATRP initiator containing a 2-bromoisobutyryl group. The glass plate modified with a PCMB brush was highly hydrophilic and showed a strong resistance against non-specific adsorption of proteins and cell adhesion. Upon ion beam irradiation, furthermore, the PCMB brush was ablated and a hollow space with a designed shape could be made to which HEK293 cells (from human embryonic kidney) and Hep G2 (from human hepatoma) cells non-specifically adhered, while no adhesion of these cells to the non-treated area on the brush was observed. The present results clearly indicate the usefulness of ion beam-printed patterns of anti-biofouling zwitterionic polymer brushes in the biomedical field.

Facile fabrication of uniform size-controlled microparticles and potentiality for tandem drug delivery system of micro/nanoparticles

This article describes a rapid and facile method for manufacturing various size-controlled gel particles with utilizing inkjet printing technology. Generally, the size of droplets could be controlled by changing nozzle heads of inkjet printer, from which ink solution is ejected. However, this method uses drying process before gelling microparticles, and with that, the size of microparticles was easily controlled by only altering the concentration of ejected solution. When sodium alginate solution with various concentrations was ejected from inkjet printer, we found that the concentration of alginate solution vs. the volume of dried alginate particle showed an almost linear relationship in the concentration range from 0.1 to 3.0%. After dried alginate particles were soaked into calcium chloride solution, the size of microgel beads were obtained almost without increasing their size. The microparticles including various sizes of nanoparticles were easily manufactured by ejecting nanoparticle-dispersed alginate solution. The release of 25-nm sized nanoparticles from alginate microgel beads was finished in a relatively-rapid manner, whereas 100-nm sized nanoparticles were partially released from those ones. Moreover, most of 250-nm sized nanoparticles were not released from alginate microgel beads even after 24-h soaking. This particle fabricating method would enable the tandem drug delivery system with a combination of the release from nano and microparticles, and be expected for the biological and tissue engineering application.

3D Micro-fabrication by Inkjet 3D biofabrication for 3D tissue engineering

3D micro-fabrication technique is one of the essential technologies in manufacturing complicated 3D biological tissues by science and technology. We have developed inkjet 3D biofabrication approach as a technology to build up designed 3D biological tissues with micro- to macro-structures by handling different multi-types of living cells. In our technique using inkjet printing technique with gel precursor and gel reactant, 3D hydrogel structures can be fabricated. As a biocompatible hydrogel, we use alginate hydrogel, which protects living cells from drying and keep 3D structures in biological liquid media. With a custom made 3D bioprinter, several 2D and 3D structures have ever been fabricated. In this study, we tried to fabricate more miniaturized 3D structures. Using new version of experimental inkjet nozzle system, miniaturized 3D hydrogel tube could be successfully fabricated, which diameter and wall thickness were 100 mum and 40 mum, respectively. Compared to the previous gel tube of 1 mm diameter, substantial size reduction was achieved. Drop on demand inkjet 3D biofabrication approach has a good potential towards manufacturing of micro-scaled 3D tissue structures, which is essential structure for complicated biological tissues.

Biofabrication: A Guide to Technology and Terminology

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.